Chemoenzymatic synthesis of prodigiosin analogues exploring the substrate specificity of PigC

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1 Chemoenzymatic synthesis of prodigiosin analogues exploring the substrate specificity of PigC Suresh R. Chawrai, eil R. Williamson, George P. C. Salmond and Finian J. Leeper Representative Experimental Details Supplementary Material B(O) 2 O DEF/POBr Br 75% [Pd(PPh ) ]cat. 6 79% 7 8a CO -((5-Bromo--methoxy-2-pyrrol-2-ylidene)methyl)--ethylethanamine 1 7 To a mixture of diethylformamide ( equiv, 0. ml) and DCM (0.5 ml) at 0 o C was added dropwise a solution of phosphorus oxybromide (2.5 equiv, 0.6 g) in DCM (1.5 ml). The mixture was stirred at 0 o C for 0 min and then evaporated to dryness in vacuo. DCM (1.0 ml) was added to the solid and cooled to 0 o C. A solution of -methoxy--pyrrolin-2-one (0.1 g, 0.88 mmol) in DCM (2.0 ml) was added dropwise. The mixture was warmed to room temperature, then heated at 0 o C for 0 min, then stirred at room temperature for h and finally poured onto aqueous sodium bicarbonate-ice-etoac mixture and stirred for 10 min. The resulting mixture was filtered through Celite and the two layers were separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO, filtered and evaporated under reduced pressure. The residue was purified by column chromatography (silica gel neutralized with 0.75% TEA; eluent, EtOAc:petroleum ether, 1.5:10) to obtain the enamine 7 as an oil (170 mg, 75%), which turned into to a crystalline solid when kept in a refrigerator, m.p. 8 0 o C (Found: C, 6.25;, 5.8;, Calc. for C Br 2 O: C, 6.5;, 5.8;, 10.8%); R f 0.70 (MeO:DCM; 0.7:9.); ν max (neat) cm 1 160s (C=C); δ (00 Mz, CDCl ) 1.27 and 1.28 (each, t, J 7.2, C 2 C ),.7 and.10 (each 2, q, J 7.1, C 2 C ),.7 (, s, OC ), 5.57 (1, s, C=C- OC ), 6.98 (1, s, C=C-); δ C (100 Mz, CDCl ) and 1.82 (C C 2 ),.76 and 51.1 (C C 2 ), (OC ), (C=C-O), (C=C), 1.96 (BrC), (C=C), (C-OC ); RMS, m/z found 259.0, C Br 2 O requires M

2 -Methoxy-5-phenyl-1-pyrrole-2-carbaldehyde 8a 5 2 CO To a degassed solution of toluene (0.5 ml) were added Pd(OAc) 2 (09 g, 8 mmol) and PPh (6 g, 0.17 mmol). The mixture immediately became dark yellow and was stirred at 70 o C for 20 min under argon. A solution of bromoenamine 7 (0.1 g, 0.87 mmol) and benzeneboronic acid (71 g, mmol) in 5% water/dioxane (1.5 ml) was degassed and purged with argon. The suspension of Pd(PPh ) in toluene was added to this solution and solid a 2 CO (0.12 g, 1.16 mmol) was added. The mixture was stirred at 98 o C for 90 min and then poured into water. The p of the mixture was lowered to p 7 with M hydrochloric acid and it was filtered through Celite. The Celite was washed several times with acetone and the washings were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel neutralized with 0.75% Et (eluent, EtOAc:petroleum ether, 2:10) to give the phenylpyrrole 8a as pale yellow solid (95 mg, 79%), m.p: o C (Found: C, 71.;, 5.5;, 6.9. Calc. for C O 2 : C, 71.6;, 5.5;, 6.95%). R f 0.51 (MeO:DCM; 0.7:9.); ν max (neat) cm 1 28s (-), 2822m (aldehyde C-), 1616s (C=O); δ (00 Mz, CDCl ).92 (, s, OC ), 6.19 (1, d, J, -), 7.6 (1, tt, J 7 and 2, p-), 7. (2, t, J 7, m-), 7.55 (2, dd, J 7 and 2, o-), 9.0 (1, br s, ), 9.55 (1, s, CO); δ C (100 Mz, CDCl ) 58.2 (OC ), 9.5 (C-), (C-2), 125. (2 x o-c), (p- C), (2 x m-c), 10.7 (phenyl-c), 19.0 (C-5), (C-), (CO); RMS, m/z , C O 2 requires M Methoxy-5-thien-2-yl-1-pyrrole-2-carbaldehyde 8b ' 5' ' S 2' 5 2 CO The thienylpyrrole 8b was obtained as a brick-red coloured solid (71 mg, 88%), m.p o C (dec.); R f 0.52 (MeO:DCM; 0.7:9.0); ν max (neat) cm 1 207s (-), 2826m (aldehyde C-), 1615s (C=O); δ (00 Mz, CDCl ).90 (, s, OC ), 6.08 (1, d, J 2, -), 7.07 (1, dd, J 5 and, -), 7.0 (1, dd, J and 1, 5 -), 7.2 (1, dd, J 5 and 1, -), 9.07 (1, bs, ), 9.52 (1, s, CO); δ C (100 Mz, CDCl ) 58.2 (OC ), 9.9 (C-), (C-2), 12.95, and ( x thienyl-c), 1.86 and 1.97 (C-5 and C-2 ), (C-), (CO); (Found: C, 5.9;,.;, 5.9. Calc. for C 10 9 O 2 S: C, 57.95;,.;, 6.8%); RMS, m/z , C 10 9 O 2 S requires M

3 -Methoxy-5-fur-2-yl-1-pyrrole-2-carbaldehyde 8c ' 5' ' O 2' 5 2 CO The furylpyrrole 8c was obtained was obtained as golden coloured solid (5 mg, 95%), m.p.: 1-16 o C. R f 0.52 (MeO:DCM; 0.7:9.0); ν max (neat) cm 1 207s (-), 2826m (aldehyde C-), 1616s (C=O); δ (00 Mz, CDCl ).8 (, s, OC ), 6.0 (1, s, -), 6.2 (1, dd, J and 2, -), 6.6 (1, dd, J and 1, -), 7.9 (1, dd, J 2 and 1, 5 -), 9.0 (1, br s, ), 9.7 (1, s, CO); δ C (100 Mz, CDCl ; due to low solubility only carbons with protons attached gave visible peaks) 58.2 (OC ), 92. (C-), (C- ), (C- ), 1.1 (C-5 ), 17.8 (CO). LCMS, m/z 192, C 10 9 O requires M Methoxy-5-indol-2-yl-1-pyrrole-2-carbaldehyde 8d 5' 6' ' 7' ' 2' 5 2 CO In this case the starting boronic acid was the -Boc derivative of indole-2-boronic acid and two products were obtained, the indolylpyrrole 8d and its indolyl--boc derivative in a combined yield of 96%. Indolylpyrrole 8d was obtained as a colourless solid (1 mg, 28%), m.p o C (dec.). R f 0.26 (EtOAc:exane; 1:1); ν max (neat) cm 1 20s (-), 285m (aldehyde C-), 158s (C=O); δ (00 Mz, CD OD; COSY used for assignments).97 (, s, OC ), 6. (1, s, -), 6.97 (1, s, -), 7.05 and 7.17 (each 1, td, J 8 and 1, 5 - and 6 -), 7.9 and 7.57 (each 1, dd, J 8 and 1, - and 7 -), 9.8 (1, s, CO); LCMS, m/z 21, C O 2 requires M Chromatography also gave the indolyl--boc derivative (5 mg, 68%). R f 0.5 (EtOAc:exane; 1:1); ν max (neat) cm 1 20s (-), 285m (aldehyde C-), 158s (C=O); δ (00 Mz, CD OD).96 (, s, OC ), 6.20 (1, s, -), 6.88 (1, s, -), 7.27 and 7.7 (each 1, td, J 8 and 1, 5 - and 6 -), 7.61 and 8.22 (each 1, dd, J 8 and 1, - and 7 -), 9.5 (1, s, CO); δ C (100 Mz, CD OD) 27.0 (CMe ), 57.7 (), 8.2 (CMe ), 96. (C-), 112. (C- ), 115. (C-7 ), 119. (C-5), (C-5 ), 12. (C- ), 125. (C-6 ), and 10.8 (C-2 and - a ), 18.0 (C-2), 19.9 (-C=O), (C-), 17.8 (CO); LCMS, m/z 1, C O requires M Methoxy-5-naphth-2-yl-1-pyrrole-2-carbaldehyde 8e 6' 5' 7' 8' ' ' 2' 5 2 The naphthylpyrrole 8e was obtained as colourless solid (79 mg, 81%), m.p.: o C. R f 0.55 (MeO:DCM; 0.7:9.0); ν max (neat) cm 1 25s (-), 2818m (aldehyde C-), 1620s (C=O); δ (00 Mz, CD OD).00 (, s, OC ), 6.62 (1, s, -), 7.56 (2, m, 6 - and 7 -), (, m, -, -, 5 - and 8 -), 8. (1, s, 1 -), 9.7 (1, s, CO); δ C (100 Mz, CD OD; due to 1' CO

4 low solubility only carbons with protons attached gave visible peaks) 57.8 (), 9.8 (C-), 12.5 (C- ), 12.8 (C-1 ), and (C-6 and -7 ), 127.9, and (C-, -5 and -8 ), 17.7 (CO); LCMS, m/z 252, C 16 1 O 2 requires M Methoxy-5-p-biphenyl-1-pyrrole-2-carbaldehyde 8f '' 2'' ' 2' 1'' ' 1' '' 5'' 6'' 5' 6' 5 2 CO The biphenylpyrrole 8f was obtained as colourless solid (88 mg, 82%), m.p.: 2-27 o C (dec.). R f 0.57 (MeO:DCM; 0.7:9.0); ν max (neat) cm 1 29s (-), 2826m (aldehyde C-), 162s (C=O); δ (00 Mz, DMSO).90 (, s, OC ), 6.6 (1, d, J 2, -), 7.9 (1, tt, J 1 and 8, -), 7.9 (2, t, J 8, and 5 -), (, m,, 5, 2 and 6 -), 8.00 (2, d, J 8, 2 and 6 -), 9.7 (1, s, CO), (1, br s, ); δ C (100 Mz, DMSO; MQC used for assignments) 58. (OC ), 9.2 (C-), (C-2), (C-2 and 6 ), and 127. (C-, 5, 2 and 6 ), (C- ), 129. (C- and 5 ), 10.1 (C-1 ), 18.1 (C-5), 19.7 and 10.1 (C- and 1 ), (br, C- ), 17.8 (CO); LCMS, m/z 278, C O 2 requires M Feeding Experiments (a) On agar plates Streaks of the following Serratia mutants were grown for two days on PGM-agar plates: pig57, pigm, pigb, pigc, and pigd. Beside each streak was spotted on the agar 10 µl of a DMSO solution (0, 1, or 10 mm) of MBC 1 or one of its analogues 8a-f. The plate was then incubated at 0 o C for 8 h before being photographed, as shown below. The photographs shown are of the pig57 strain. The pigm strain gave very similar results. o pigmentation was observed when 1 or 8a-f were fed to either of the mutants, pigb or pigd, blocked in the biosynthesis of the monopyrrole MAP 2 or to the pigc mutant, which is defective in the production of the condensing enzyme.

5 Fig. S1. Feeding of MBC 1 and its analogues to streaks of the Serratia pig57 strain grown on agar plates. (i) MBC 1 (ii) Phenyl analogue 8a (iii) Thienyl analogue 8b (iv) Furyl analogue 8c (v) Indolyl analogue 8d (vi) aphthyl analogue 8e

6 Fig. S1 (continued). (vii) Biphenyl analogue 8f (b) In liquid culture Cells were grown in LB broth (Tryptone 10 g/l, yeast extract 5 g/l, acl 5 g/l) supplemented with sorbitol (0.25 M) for Serratia strains or ampicillin (100 µg/ml) for E. coli strains. Flasks were inoculated with an appropriate starter culture to get an OD 600 of and then grown in the shaker for h at 0 o C for Serratia strains or at 7 o C for E. coli strains. Sufficient solution of MBC 1 or one of its analogues 8a-f (10 mm in DMSO) was added to give the final concentrations shown in Table 1 of the paper. The cultures were then shaken for a further h, to an OD 600 of about 0 for the Serratia strains or about - for the E. coli strains. Each culture was centrifuged and the cell pellet was extracted with ethanol acidified with % of 1 M hydrochloric acid (1 ml) and then centrifuged again. A UV/Vis spectrum was recorded for the ethanolic supernatant (with appropriate dilution when the absorbance exceeded 1) and it was then evaporated to dryness and analysed by LC-MS. Cuvettes from the Serratia pig57 strain incubated with 100 µm MBC or analogue are shown in the paper, Fig. 1. Fig. S2. Cuvettes from the Serratia pig57 strain incubated with 10 µm MBC 1 or analogues 8a-f.

7 Fig. S. Selected chromatograms from the LC-MS analysis of the extracts (a) Extract of medium from culture of E. coli strain D5α-pRW7 supplemented with MBC 1; selective ion chromatogram for m/z = 2 (M+ + for prodigiosin ). F_Leeper806-1 Sm (Mn, 1x2) % (b) Extract of medium from culture of E. coli strain D5α-pRW7 supplemented with phenyl analogue 8a; total ion chromatogram; the peaks labelled 8a and 9a can be identified by their mass spectrum as corresponding to those compounds. SC-051-PrG-benz F_Leeper e F_Leeper806-1 Sm (Mn, 1x2) : Diode Array Range: 8.55e+1 6.0e+1.0e a.66 9a e F_Leeper80-1 Sm (Mn, 1x2) 1: Scan ES- (c) Extract of medium from culture of E. coli strain D5α-pRW7 supplemented with thienyl analogue 8b; total ion chromatogram; the peaks labelled 8b and 9b correspond to those compounds. F_Leeper : Diode Array Range: 1.019e+2 8.0e+1 6.0e b 9b.19.0e e F_Leeper805-1 Sm (Mn, 1x2) 2: Scan ES+

8 Fig. S (continued). (d) Extract of medium from culture of E. coli strain D5α-pRW7 supplemented with furyl analogue 8c; total ion chromatogram; the peaks labelled 8c and 9c correspond to those compounds. sc-052-prg-furan F_Leeper e+2 8.0e+1 6.0e+1.0e c.1 9c.01 : Diode Array Range: 1.262e+2 2.0e F_Leeper80-1 Sm (Mn, 1x2) 2: Scan ES (e) Extract of medium from culture of Serratia mutant pig57 supplemented with indolyl analogue 8d; selective ion chromatogram for m/z = 7 (M+ + for prodigiosin analogue 9d). F _ Le ep e r S m (M n, 1 x2 ) 2 : S c an E S e 6 9d (f) Extract of medium from culture of Serratia mutant pig57 supplemented with indolyl analogue 8d and 2,-dimethylpyrrole 10; selective ion chromatogram for m/z = 17 (M+ + for obatoclax 5). s c In d o ly l- D M P -a f t e r e v a p F _ L e e p e r S m ( M n, 1 x 2 ) 2 : S c a n E S e 6 % % F _ Le ep e r S m (M n, 1 x2 ) 2 : S c an E S (g) Extract of medium from culture of E. coli strain D5α-pRW7 supplemented with naphthyl analogue 8e; total ion chromatogram; the peak labelled 8e corresponds to that compound; no peak was found for 9e. F_Leeper e e p e m ( M c a n F _ L r S n, 1 x 2 ) 2 : S E S e+2 2.5e e : Diode Array Range:.22e+2 2.0e+2 1.5e e F_Leeper88-1 Sm (Mn, 1x2) 1: Scan ES-

9 Fig. S (continued). (g) Extract of medium from culture of E. coli strain D5α-pRW7 supplemented with biphenyl analogue 8f; total ion spectrum; the peak labelled 8f corresponds to that compound; no peak was found for 9f. F_Leeper f : Diode Array Range: 2.02e+2 1.5e e F_Leeper89-1 Sm (Mn, 1x2) 1: Scan ES- (h) Extract of medium from culture of E. coli strain D5α-pRW7; total ion spectrum; the peak labelled 2 corresponds to MAP. F_Leeper e e+2 7.5e+1 5.0e : Diode Array Range: 1.666e+2 2.5e F_Leeper818-1 Sm (Mn, 1x2) 1: Scan ES